Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes serious illness in pregnant women, neonates, and immunocompromised individuals. Bacterial infections are generally food- borne and listeriosis is the leading cause of death from contaminated food products. L. monocytogenes serves as an excellent model system for exploring the interactions that take place between an intracellular parasite and its host. The overall goal of this proposal is to define the mechanisms by which L. monocytogenes senses its different host cell compartmental environments and responds with the regulated expression of gene products required for virulence. In Aim I, the temporal expression of virulence genes of individual bacteria within different host cell compartmental environments will be measured. Bacterial gene fusions to the bioluminescent green fluorescent protein (GFP) gene of Aequorea Victoria will be constructed and introduced into the L. monocytogenes chromosome. Macrophage cells infected with the L. monocytogenes gfp fusion mutants will be monitored for intracellular bacterial gene expression using fluorescent microscopy. These experiments will determine which bacterial gene products are expressed in specific host cell environments. In addition, L. monocytogenes mutants with altered patterns of intracellular gene expression will be isolated and characterized. It has been previously demonstrated that the expression of L. monocytogenes virulence determinants is regulated by a key transcriptional activator protein, PrfA. A model has been proposed that suggests that the relative concentrations of PrfA within the bacterial cell govern which virulence genes are expressed. In Aim II, the relative affinity of PrfA for its target promoters will be determined using gel mobility shift and foot printing assays, as well as a Bacillus subtilis-based expression system which enables characterization of promoter mutants. These experiments will assess if the DNA binding affinity of PrfA for a promoter determines the temporal expression and the level of transcriptional activation of that gene. In Aim III, additional L. monocytogenes gene products subject to PrfA regulation will be identified and characterized. These gene, products may represent additional virulence determinants that contribute to L. monocytogenes pathogenesis.